Opthalmological collagen coverings

ABSTRACT

Ophthalmologic collagen coverings derived from animal eyes and a process   preparing such coverings comprising treatment of a fibrous tunic of an animal eye with an alkali metal hydroxide in a saturated solution of a salt of an alkali metal, followed by neutralization, dissolution in an aqueous solution of an organic acid, purification of the resulting solution of collagen by dialysis against a buffer solution while bringing the solution of collagen to a pH of 4.5 to 7.5 and drying the thus-obtained solution of collagen simultaneously with shaping spherical coverings therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 461,172,filed 1/5/90, abandoned, which is a divisional of U.S. Ser. No. 131,958,filed 12/11/87, U.S. Pat. No. 4,913,904, which is a continuation-in-partof U.S. Ser. No. 908,349, filed 9/17/86, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a form of collagen that is uniquely suited forophthalmic uses. In more specific embodiments, this invention relates toa process for preparing ophthalmological collagen coverings and tocoverings prepared by this process and useful in all kinds ofophthalmological operations, as well as in a conservative treatment oftraumatic and trophic injuries of the cornea of the eye having differentetiology.

The present stage of the development of eye surgery features a highquality of surgical treatment. In this respect there is a problem ofdeveloping new methods and agents making it possible to reduce thenumber of post-operational complications, to shorten the time ofhospital stays, and to ensure higher functional results of operations onthe globe of the eye.

Known in the art are materials produced from collagen and methods ofdissolution of collagen derived from the skin and tendons of animals.Thus, a known process for dissolution of collagen of an animal skin(USSR Inventor's Certificate No. 162280, 1964) comprises an alkali-salttreatment of an animal skin with a subsequent neutralization andresidence in an acidic medium to give a dispersion of collagen having pHof not higher than 2.5.

Known in the art is a process for preparing a hemostatic material from adispersion of collagen (cg. USSR Inventor's Certificate No. 561564,1977). Acidification of neutral dispersions of collagen obtained afteran intensive alkali-salt treatment of animal skin and tendons areintermixed with thrombin and antibiotics, where after the mixture isfrozen and dried in vacuum.

These processes cannot be employed for the preparation of collagencoverings to be used in ophthalmology, since the materials produced bythese processes have clearly pronounced antigenic properties.

It is an object of the present invention to provide a process forpreparing and using ophthalmological collagen coverings. A particularobject, is a covering for use in connection with microsurgicaloperations on the globe of the eye. A further object is an improvedconservative treatment of traumatic and trophic injuries of the corneaof the eye having different etiology. Other objects will be apparentfrom consideration of the following description of the invention.

SUMMARY OF THE INVENTION

It has been found that collagen isolated from animal eyes, especiallythe fibrous tumic of animal eyes and preferably the sclera of animaleyes, is specially suited for topical application to the eye, avoidingantigenic characteristics associated with collagen derived from, e.g.,animal dermis. The collagen also possesses therapeutic properties makingit desirable for the treatment of traumatic and trophic injuries of thecornea. The collagen further possesses lubricating properties suggestingthe potential for the treatment of dry eye.

The collagen may be provided in a variety of forms adapted forophthalmic use. Examples include membranes, gels, and solutions.Especially preferred for the treatment of traumatic and trophic injuriesare shaped collagen articles (described in more detail below). Collagenapplied to the eye in the form of, e.g., drops or ointments is washedrelatively rapidly from the eye by tear flow. By contrast, collagenapplied as a shaped article is maintained in the eye for a longerperiods, extending the desirable therapeutic effects of the application.Even when applied as a shaped article, however, the collagen isbioerodible and will dissolve in the eye.

The preferred process for isolating collagen from animal eyes and forpreparing ophthalmological collagen coverings comprises treating thefibrous tunic of an animal eye with an alkali metal hydroxide in asaturated solution of an alkali metal salt, then neutralizing theresulting tissue to a pH of from 6.0 to 7.0, and dissolving the tissuein an aqueous solution of an organic acid; the thus-prepared solution ofcollagen is decontaminated from low-molecular impurities by dialysisagainst a buffer solution while bringing pH of the solution of collagento 4.5-7.5 and the obtained solution of collagen is dried simultaneouslywith shaping therefrom spherical coverings repeating the curvature ofthe front section of the eye.

With the view to improve quality of the resulting covering, as thestarting material--fibrous tunic of an animal eye use is preferably madeof the sclera of animal eyes.

The neutralization of the tissue should be preferably effected by meansof a 2% solution of boric acid. For dissolution of collagen any organicacid can be used, acetic acid being preferable. The ophthalmologicalcollagen coverings prepared by the process according to the presentinvention has a spherical shape repeating the curvature of the frontsection of the eye and ensuring a full contact with the surface of thecornea of the eye; they also have dimensions ensuring a full coating ofthe cornea.

The ophthalmological collagen coverings according to the presentinvention are applied onto the front surface of the cornea and serve asa temporary hydrophilic spherical "bandage" and kept on the frontsurface of the cornea without any additional fixation while exhibitingtherapeutical properties of a naturally-occuring copolymer--collagen.

The coverings according to the present invention make it possible toreduce the number of post-operational complications, accelerate healingof injured tissues of the eye. In the case of traumatic and trophicinjuries of the cornea the collagen coverings according to the presentinvention enable normalization of metabolic processes in the injuredcornea. The coverings of this invention are useful in the front radialkeratotomy, especially in the case of appearance of microperforations,in keratoplasty, keratoprosthetics, in the treatment of post-operationalkeratopathy, endothelial-epithelial dystrophy, erosion of the cornea.The use of ophthalmological collagen coverings makes it possible toreduce the number of post-operational complications, in 98% of casesthere has been noted the formation of more soft cicatrices on the corneaafter keratotomy; in 98% of cases there has taken place a lesser degreeof edema of the cornea and the transplantate edema in keratoplasty. Introphic injuries of the cornea having different etiology positivedynamics has been noticed in 95% of cases as compared to previouslycarried out conservative therapy with the use of pharmaceuticalpreparations.

DETAILED DESCRIPTION OF THE INVENTION

The source of the collagen is mammalian eyes, preferably the eyes ofanimals, and most preferably agricultural animals, such as pigs. Thecollagen may be extracted from any collagen containing, discrete portionof the eye or from the whole intact eye, but the preferred source ofcollagen is from the sclera of the eye. Collagen isolated from animaleyes and used in ophthalmological products according to this inventioncauses minimal allergic reaction and less eye inflammation as comparedto collagen isolated from other sources, such as bovine hide.

The fibrous tunic of an animal eye is thoroughly cleaned from internalcoats of the eye, residual epithelium, conjunctiva, muscles andmiscellaneous; and stroma is isolated.

The isolated stroma is cut into small pieces. The cut tissue is rinsedwith distilled water to completely remove mechanical foreign matter andblood, then it is transferred into a flask and to which is then added analkali metal hydroxide in a saturated solution of a salt of an alkalimetal, for example, with a 10% solution of caustic soda in a saturatedsolution of sodium sulphate (at a rate of 500 ml of the solution per 10g of the tissue) for 48 hours at a temperature of 18°-25° C. Thesolution is decanted and the tissues are subjected to neutralization toa pH of 6.0-7.0 under stirring, e.g. in a 2% solution of boric acid andrepeatedly changing the solution. The tissue is rinsed with distilledwater until a complete removal of the sulphate ion in the rinsing liquidThe rinsed tissue is then dissolved in a 0.5-1M solution of an organicacid such as acetic acid in such quantity that the final concentrationof protein in the solution be equal to 1%. As the organic acid use canbe made of citric acid, lactic acid, ascorbic acid and the like. Themass is stirred and allowed to stand in a refrigerator for 1-3 days at alow temperature. Then the mass is homogenized, centrifuged and allowedto stand for one day at a low temperature. The resulting solution isfiltered. To carry out neutralization and desolvation, the acetic-acidsolution of collagen is diluted with acetic acid to a concentration ofprotein of 0.7-0.8% and dialyzed against a phosphate or citrate bufferat a temperature of 18°-20° C. while bringing pH of the solution ofcollagen to 4.5-7.5. The resulting collagen solution is centrifuged,poured into matrices repeating the shape of the front section of the eyeand air-dried in a dust-free cabinet at a temperature within the rangeof from 10° to 27° C. Ophthalmological collagen coverings are thusobtained which are elastic, transparent, of a spherical shape repeatingthe curvature of the front section of the eye. The size and shape ofcoverings ensure a full coating of the cornea of the eye and its fullcontact with the front surface thereof. The final coverings aresterilized.

Preferred embodiments of the foregoing process steps are:

1. The sclera pieces are treated with an aqueous solution comprisingfrom 1.0 to 3.0 molar alkali metal hydroxide and from 0.8 to 1.5 molaralkali metal sulfate, preferably 2.5 molar sodium hydroxide and 1.4molar sodium sulfate having a pH of about 12-14. Sclera is treated withstirring for about one to three days, preferably for two days. Theweight to volume ratio of sclera to treatment solution is from about 15to 25 grams per liter, preferably about 20 grams per liter.

2. The sclera is neutralized to a pH of about 6 to 7 by draining thehydroxide solution and treating first with distilled water and then witha dilute aqueous acid solution having a pH of about 3-5, preferably 4 to4.5. Acids useful for neutralization include boric acid, tartaric acid,citric acid, acetic acid, lactic acid and ascorbic acid. Boric acid ispreferred for neutralization, with a concentration of about 0.02 to 0.04molar, preferably 0.030 to 0.033 molar. Treatment cycles are typically15 minutes to two hours, with constant stirring. The weight to volumeratio is typically 20 grams (initial starting weight) to from about 500milliliters to one liter treatment solution. Two to five acid treatmentsare used to neutralize the sclera; preferably, three treatments areused. The sclera is rinsed with distilled water followingneutralization; preferably, three times for about 1 hour each rinse.

3. The sclera is then dissolved in an aqueous organic acid solution to aconcentration of about 0.5% to 1.5% by weight collagen, preferably to aconcentration of 1%. Acids suitable for preparation of collagen solutionare the organic ones listed previously for neutralization of sclera. Thecollagen is dissolved by incubation in a suitable aqueous organic acid,preferably acetic acid, with a concentration of about 0.1 to 2 molar,preferably 1 molar, with stirring for about two to four days, preferablythree days, at a temperature of about 2 to 10 degrees centigrade,preferably 4 to 6 degrees centigrade.

4. The collagen solution is conviently homogenized in a blender,centrifuged and filtered. The collagen solution is then dialyzed againstan aqueous buffer, see e.g. U.S. Pharmacopeia XXI, 1985, page 1420, suchas phosphate or citrate, preferably citrate, having a concentration ofabout 0.002 to 0.2 molar, and a pH of about 7.0 to 8.0, preferably 7.2.Dialysis is continued with addition of fresh citrate buffer until the pHof the collagen is from about 4.5 to 7.5, preferably about 5.0 to 5.5,and the collagen solution has formed a cloudy, homogenous gel. Dialysismay be accomplished by various techniques known to the art. Typically,dialysis membranes are used having molecular weight cut-off limits offrom about 3,000 to 100,000, preferably from about 10,000 to 100,000.These membranes easily retain the extracted collagen, which has anaverage molecular weight of about 300,000. This molecular weight istypical of tropocollagen, defined herein and in the literature as thebasic molecular subunit of collagen, existing as a rigid rod consistingof three polypeptide chains wound together in a triple helix.

5. The collagen gel is conveniently homogenized in a blender,centrifuged, deaerated, and filtered to remove particulate matter fromthe gel.

The collagen of this invention can be used as a vehicle for drugdelivery. For example, shaped collagen may be impregnated with anophthalmically active drug or collagen solution or gels may be employedto produce soluble preparations for ophthalmic use. Selection of theophthalmically active drug is not critical to this invention, althoughmaterials such as pilocarpine, dexamethazone, and gentamycin may bementioned as being exemplary.

Shaped collagen articles prepared as described above have very littlecross-linking, and, consequently, dissolve in a relatively slow periodof time. Dissolution rate of the collagen may be slowed if desired byintroducing a minor amount of cross-linking into the collagen structure.This may be accomplished by any of the techniques well-known in the art.Radiation induced cross-linking is particularly convenient. However, thedegree of cross-linking, if induced is limited to that which does notundermine the bioerodible properties of the articles.

The process according to the present invention makes it possible toprepare ophthalmological collagen coverings the use of which contributesto reducing post-operational complications, accelerates healing ofinjured tissues of the eye, reduces hospital stays of post-operationaltreatment of patients. The use of coverings according to the presentinvention in the case of traumatic and trophic injuries of the corneaexerts a favorable influence on metabolic processes in the cornea whichis objectively revealed in diminished photophobia, lachrymation, edemaof the cornea, better acuity of vision. This invention, and propertiesof the products, will be better understood by reference to the followingexamples.

EXAMPLE 1

To 20 g of purified and cut stroma of sclera of an animal eye 1 liter ofa 10% solution of caustic soda in a saturated solution of sodiumsulphate is added and allowed to stand at a temperature of 18°-20° C.for 48 hours. Then the solution is decanted, the tissue is rinsed with asmall amount of distilled water, added with 1 liter of a 2% solution ofboric acid and agitated by means of a magnetic stirrer for 2 hours twicechanging the solution of boric acid. Under continuous stirring thetissue is thoroughly rinsed with distilled water (total volume ofwater--5 liters) till a complete removal of the sulphate ion from therinsing liquid. 250 ml of the resulting water-treated tissue are addedwith 350 ml of a 0.5M acetic acid, stirred and left for one day at thetemperature of 4° C. Then the mass is homogenized by means of amicrodisintegrator of tissues, centrifuged for 30 minutes at 2,000r.p.m. and allowed to stand for 3 days at the temperature of 4° C. Theresulting solution is filtered through a glass filter. The thus obtained1% solution of collagen is diluted with a 0.5M solution of acetic acidto the concentration of 0.8%. The dialysis of the 0.8% solution ofcollagen is effected against a 0.2M citrate buffer while bringing pH ofthe solution of collagen to 6.7. The dialysis is continued whilelowering the buffer concentration from 0.2M to 0.002M in 3-4 stagesusing, in the last stage, heating to a temperature of 28°-30° C. Theresulting solution of collagen is centrifuged for 15 minutes at 1,000r.p.m., poured into matrices repeating the shape of the front section ofthe eye and air-dried in a dust-protected cabinet for 48 hours at thetemperature of 15° C. and relative humidity of 40-50%. The finalcoverings are sterilized by gamma-rays in the dose of 2.5 MRad and doserate of 0.5 MRad/h.

The thus-prepared coverings are transparent, elastic; they have aspherical shape repeating the curvature of the front section of the eye.The shape and dimensions ensure a full coating of the cornea of the eyeand a full contact with the front surface thereof.

EXAMPLE 2

Twenty grams of comminuted sclera, derived from porcine eyes, weretreated in a one liter flask with 980 milliliters of a solutioncomprised of 100 grams per liter sodium hydroxide and 200 grams perliter sodium sulfate in distilled water by stirring continuously for 48hours. The solution was then drained and the sclera rinsed by a 5 minutestirring cycles with 1 liter of distilled water. The sclera was thenneutralized by three 1 hour stirring cycles, each in one liter of asolution comprised of 20 grams boric acid per liter in distilled water,followed by two 15-minute stirring cycles, each in one liter ofdistilled water. During neutralization, the sclera swells significantlyand becomes soft and transparent. The swollen sclera was then dissolvedin 530 milliliters of 1 molar acetic acid by stirring at 4 degreescentrigrade for 48 hours to form a collagen solution having anapproximate concentration of 1%. The resulting collagen solution washomogenized in a blender, centrifuged to remove air bubbles and filteredto remove fine retinal fragments. The collagen solution was then placedin dialysis tubing having a molecular weight cut-off of 10,000-12,000and dialyzed against 25 liters of a citrate buffer solution comprised of22.5 grams of sodium citrate in 25 liters of distilled water, pHadjusted to 7.2 with 1 molar hydrochloric acid. As a result of thedialysis, the collagen solution formed a cloudy, white, homogeneous geland had a pH of 5.2. The gel was then homogenized in a blender,centrifuged, deaerated and filtered.

The resulting solution was cast into molds conforming to the curvatureof the eye and allowed to dry at room temperature in a laminar flow hoodfor 72 hours. The corneal coverings have an average thickness of about0.002 inches and weigh about 0.003 grams.

COMPARATIVE EXAMPLE A

Dialysis is a critical step in the production of collagen materialsuitable for casting. When cast from acidic, predialysis solutions, theresulting collagen coverings are typically malformed with numerousinhomogeneities and defects. Because of differences in the vaporpressures of water and acetic acid, and in solutions employing otheracids, acid concentration can increase upon drying causing denaturationof the collagen. As an example, collagen derived as described in Example1 but only dialyzed against 10 liters of buffer, has a pH of 3.5 andwill not cast clear, homogeneous films, free of imperfections.

EXAMPLE 3

The significance of the use of the collagen covering disclosed hereinfollowing ophthalmic surgery or corneal trauma has been demonstrated inexperiments in rabbits. Rabbits were subjected to standard radialkeratotomy procedures in both eyes, and one eye was covered with acollagen covering immediately following surgery. Examination at varioustimes following surgery showed a marked decrease in traumaticinflammation and edema at all time following surgery for the eyestreated with the coverings. It was also observed that incisions withoutthe covering widened as they healed, filling with a wedge of epitheliumwhile those treated with collagen covering were held together and"bridged" by the covering. Fibroblast proliferation at the site was twoto three times greater than the controls indicating higher levels ofcollagen synthesis. The coverings dissolved within two to six hours.

It has also been found that if collagen other than that derived from theeye, such as bovine dermal collagen, is used to cover ophthalmic wounds,inflammation and edema are more severe than for porcine eye collagen.Further, collagen from the eyes of pigs elicits somewhat lessinflammatory response than from the eyes of cows.

The advantages of this invention will be apparent to those skilled inthe art. Greatly improved, virtually non-allergic products areeconomically made available for the healing arts.

It will be understood that this invention is not limited to the specificexamples which have been offered as particular embodiments, and thatmodifications can be made without departing from the spirit thereof.

What is claimed:
 1. An ophthalmic composition comprising bioerodiblecollagen isolated from the sclera of eyes and an ophthalmically activedrug.
 2. Collagen isolated from the sclera of animal eyes by a methodwhich comprises:(a) treating the sclera of an animal eye with an alkalimetal hydroxide in a saturated solution of an alkali metal salt, (b)neutralizing the resulting collagen tissue to a pH of 6.0-7.0, (c)dissolving the collagen tissue in an aqueous solution of an organicacid, and (d) dialyzing the resulting collagen solution against a buffersolution to raise the pH of the collagen to 4.5-7.5.